Consolidation of the Thioredoxin Fold by Peptide Recognition: Interaction between E. coli Thioredoxin Fragments 1-93 and 94-108

SANTOS J; MARINO-BUSLJE C; KLEINMAN C; ERMACORA M; DELFINO JM

BIOCHEMISTRY

Año: 2006 vol. 46

0006-2960

Escherichia coli thioredoxin (TRX) catalyzes redox reactions via the reversible oxidation of the conserved active center WCGPC. TRX is a monomeric R/ protein with a fold characterized by a central -sheet surrounded by R-helical elements. The interaction of the C-terminal R-helix (helix 5) of TRX against the remainder of the protein involves the close packing of hydrophobic surfaces, opening the possibility of studying a fine-tuned molecular recognition phenomenon. To evaluate the relevance of this interaction on the folding mechanism of TRX, we characterize TRX1-93, a truncated variant of TRX devoid of the last stretch of 15 amino acid residues that includes helix 5. TRX1-93 may possibly represent a molecular form where the folding process becomes interrupted, giving rise to a structure exhibiting the features of a molten globule state. This was assessed by circular dichroism, intrinsic fluorescence, binding of the probe ANS, size-exclusion chromatography, limited proteolysis, and calorimetry. Remarkably, fragment TRX1-93 interacts with peptide TRX94-108 (KD ¡­2-12 ¥ìM), bringing forth the restoration of native-like signatures and enzymic function. This represents a molecular event of reciprocal structure selection where both partners gain order, thus leading to long-range consequences on conformation. In this context, the binding of the C-terminal helix could signify a late event in the consolidation of the overall TRX fold.